Peptide-linked drug delivery system

ABSTRACT

The present disclosure relates to a systemically administered peptide delivery platform that biodistributes to the kidney or urinary tract. The disclosure further relates to methods of treating a disease of the kidney or urinary tract in a subject in need thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. Pat. Application No.17/556,714, filed Dec. 20, 2021, which claims the benefit of U.S.Provisional Application Nos. 63/128,509, filed Dec. 21, 2020, and63/254,754, filed Oct. 12, 2021, the contents of each of which are fullyincorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant CA222802awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Diseases of the kidney and urinary tract cause significant morbidity andmortality. For example, urothelial carcinoma, the most common type ofbladder cancer (BC), is one of the leading cancers in the United States.Most BCs are non-muscle invasive and superficial in nature. The standardtreatments involve instillation of chemotherapeutic/immunotherapeuticagents in the bladder after initial surgical resection of the tumor.These drugs are often administered via catheterization of the urethrainto the bladder, also known as intravesical therapy (ITT). Despiteevidence of ITT’s clinical efficacy, disease recurrence rate remainshigh, up to 50%. This can be caused by incomplete drug delivery, as theadministered drug can only be retained in the bladder for a limitedtime. Furthermore, urothelial carcinoma can recur throughout the entireurothelium (renal pelvis, ureter, and bladder). Since ITT only deliversdrugs to the bladder, any tumors in the ureter or renal pelvis cannot bereached. ITT also requires invasive catheterization, which can causepain, infection, urinary symptoms, poor patient compliance, andultimately lead to treatment discontinuation. There remains a need toidentify systems that overcome the drug delivery barriers of current ITTfor treatment of diseases of the kidney and urinary tract withoutrequiring an invasive procedure or surgery. Such systems should deliversystemically administered therapies to the urinary system withoutsystemic toxicity and result in prolonging the contact time between thedrug and the urinary system, providing a more effective treatment fordiseases such as urothelial carcinoma.

SUMMARY OF THE INVENTION

In certain embodiments, the present disclosure provides a compoundrepresented by formula (I):

or a pharmaceutically acceptable salt thereof, wherein the peptide is apeptide targeted for renal clearance.

In certain aspects, the present disclosure provides a method of treatingcancer, comprising administering to a patient in need thereof a compoundor composition of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-H Bdd shows remarkable urinary disposing properties. (FIG. 1A)Conventional intravesical chemotherapy (ITC) is invasive, and the drugdelivery is limited to bladder. Bdd is designed to carry multiplenegative charges that minimize off-targeting delivery and facilitatesrenal excretion after intravenous (i.v.) administration. When used as adrug carrier, Bdd promotes drug deposition into the entire urothelialsystem and provides a continuous drug excretion for a more comprehensivetreatment. (FIG. 1B) A table showing the components and the net chargesof BDD as well as its D-configuration (Bdd), neutrally charged (BKD),positively charged (BKK), and pegylated (PEG₃DD) counterparts. (FIG. 1C)A synthetic scheme showing radiolabeling of the peptides with ⁸⁹Zr forcomparative PK and biodistribution studies. (FIG. 1D) RepresentativeµPET/CT images of BALB/c mice acquired 1, 4, and 24 h afteradministration of different ⁸⁹Zr-radiolabeled peptides (20 µCi, 20 µg,in 100 µL PBS) via tail-vein injections (n=4/group). The whole-bodyimages were acquired with or without emptying the animal’s bladders.(FIG. 1E) Bar chart showing the amount of radioactivity in urine samples(20 µL) collected from animals (n=5) at different time intervals afteradministration of ⁸⁹Zr-Bdd. (FIG. 1F) Comparing the renal clearance ofthe radiolabeled peptides. Plots showing the percentage of injected dose(% ID) in kidneys (per cc) of the animals over time. The percentage ofID/cc was calculated according to the radioactivity measured at theregion of interest (ROI) of the acquired PET images. (FIG. 1G) Comparingthe pharmacokinetic (PK) profiles of the radiolabeled peptides(n=4/analogue). Blood samples (20 µL) were collected at various timeintervals after injection of the peptides or free ⁸⁹Zr to the animals.The results (radioactivity measured in blood samples) were fit into atwo-compartmental model for determining the t_(½α)(half-life ofdistribution phase) and t_(½β) (half-life of elimination phase). Theother PK parameters are available in FIG. 7A. (FIG. 1H) End-pointbiodistribution study of ⁸⁹Zr-Bdd. The animals were euthanized atdifferent time intervals (n=4/time point) after administration of thepeptide (20 µCi, 20 µg, in 100 µL PBS). The amount of peptide(radioactivity) in the harvested organs were determined. The results(corrected from decay) were expressed as % ID. (Student’s t-test;**p<0.01, and ***p<0.001).

FIGS. 2A-E. Bdd effectively distributes the conjugated Cyanine5.5fluorophore to the urinary system. (FIG. 2A) A synthetic scheme of theCyanine5.5-labeled peptide (Cy-peptide) analogues. (FIG. 2B)Representative merged fluorescence/white light images of SCID miceacquired 1 and 4 h after tail-vein injection of the different Cy-peptideanalogues (0.5 nmol, 150 µL) or free Cyanine5.5 (n=4/group). (FIG. 2C)Plots comparing the amount of fluorophore in urine (% of injected dose)based on the measured fluorescence. (Lower panel) Fluorescence image ofthe urine samples (20 µL) collected from animals 1 h after the peptideor fluorophore administration. (FIG. 2D) Representative ex vivo mergedfluorescence/bright light images of the organs harvested from animals 4h after tail-vein injection of different Cy-peptides (n=4/group). (FIG.2E) Bar chart comparing peptide distribution in the harvested organs(n=4/group), based on the total fluorescence intensity. (Student’st-test; *p<0.05, **p<0.01, and ***p<0.001).

FIGS. 3A-I. Bdd as a carrier of chemotherapeutics. (FIG. 3A) Bdd doesnot trigger any innate immune response. No increase in the inflammatorycytokines concentrations was detected in the plasma of female BALB/cmice (n=3/group) 24 h after i.v. administration of the Bdd peptide (5mg/kg). LPS was used as a positive control and concentrations of eachcytokine was measured by ELISA kit. (FIG. 3B) Cellular uptake ofCyanine5.5-labeled Bdd (Cy-Bdd). Representative fluorescence microscopicimages of human UMUC-3 BC cells and murine Renca renal adenocarcinomacells incubated for 6 and 24 h with Cy-Bdd (0.5 nmol). Dapi (9 µM) andLysoTracker-GFP (1 µM) were used for nuclear (blue) and organelle(green) staining, respectively, and were added to the cells 30 min priorto imaging. Scale bar is 25 µm. (FIG. 3C) Comparing the potency ofdifferent chemotherapeutics (DM1, GEM, MIT, CIS, and DOX). UMUC-3 andRenca cells were incubated with the drugs at various concentrations for72 h prior to measuring the cell viability. The dose response curveswere plotted and the half maximal inhibitory concentrations (IC₅₀values) of each drug calculated using Graph Pad Prism 6.0 software.(FIG. 3D) Conjugation of DM1 to Bdd. The cleavable linker SPDP was firstconjugated to the peptide N-terminal in solid phase. DM1 was then addedto the cleaved peptide in a solution mixture of PBS and NMP. (FIG. 3E)Plot showing the percentage of accumulated DM1 released from the DM1-Bddover time in PBS in the absence and presence of GSH (1 mM). The amountof drug released was quantified using HPLC analysis (absorbance detectedat 254 nm). (FIG. 3F) Conjugation of aldox to Bdd. The peptide,supplemented with a N-terminal cysteine, was incubated with aldox in PBS(pH = 7.4) for 30 min prior to purification by HPLC in neutralconditions. (FIG. 3G) Plots showing the percentage of the accumulatedDOX active metabolite released from aldox-Bdd (100 µM) over time in PBSbuffers with different pH values. The amount of drug released wasquantified using HPLC analysis (absorbance detected at 480 nm). (FIG.3H) DMI--Bdd displays a similar cytotoxicity compared to free drugagainst murine bladder (MB49), human bladder (UMUC-3 and T24), andmurine kidney (Renca) cancer cell lines. Plots of relative cellviability against the drug concentration. (FIG. 3I) Aldox-Bdd is morepotent than free aldox. Plots of the relative cell viability against thedrug concentration.

FIGS. 4A-J. Therapeutic efficacy of DM1-Bdd in treating bladder cancer.(FIG. 4A) Bar chart comparing the time when BALB/c mice (n=20/group)needed to void naturally following i.v. or i.t. administration of PBS(80 µL). The bladders were emptied prior to starting the experiment.Each animal was isolated for monitoring the urination pattern. (FIG. 4B)Comparing the nephrotoxicity of DM1-Bdd to other chemotherapeutics. Barchart showing the concentrations of renal injury biomarkers, NGAL andKIM-1, in urine collected from animals 1, 3, and 7 days after treatmentwith PBS, DM1 (0.75 mg/kg), DM1-Bdd (0.75 mg/kg of drug content), MIT(0.75 mg/kg), CIS (0.75 and 10 mg/kg), or GEM (0.75 mg/kg) via tail veininjection. (Student’s t-test; *p<0.05, **p<0.01, and ***p<0.001). (FIG.4C) Immunochemical staining for NGAL and KIM-1 was also performed.Representative microscopic images of kidney sections from BALB/c micei.v. administered with PBS, DM1 (0.75 mg/kg), DM1-Bdd (0.75 mg/kg ofdrug content), or CIS (10 mg/kg) as a positive control. The organs wereharvested 3 days after the drug treatments and stained with H&E. Blackarrows indicate the multifocal degeneration of the tubular epitheliumafter treatment with CIS. Red and green arrows indicate renal tubularepithelial cells immunoreactive for NGAL and KIM-1, respectively,following CIS treatment. Scale bar is 50 µm. (FIG. 4D) Bright field andfluorescence images of UMUC-3/GFP-Luc cells that were stably transducedwith a lentivirus carrying both GFP and firefly luciferase genes. Scalebar is 80 µm. (FIG. 4E) Orthotopic xenograft model. Representative imageof bladders collected from female NSG mice (n=3) 1 week afterimplantation of UMUC-3/GFP-Luc cells (4 x 10⁴ cells/animals). Blackarrows indicate the tumors growing in the lamina propia. Scale bar is500 µm. (FIG. 4F) Representative merged bioluminescence/bright fieldimages of the tumor-bearing animals after weekly treatments with i.v.PBS (150 µL), i.v. DM1 (0.75 mg/kg, 150 µL), i.v. DM1-Bdd (0.75 mg/kg ofdrug content, 150 µL), i.t. MIT (1 mg/mL, 50 µL), i.t. DM1 (0.75 mg/kg,50 µL), or i.t. DM1-Bdd (0.75 mg/kg, 50 µL) for 3 weeks (n=10/group).Images were acquired every week to monitor and compare tumor growth ineach treatment group. (FIG. 4G) Representative pictures of bladdersexcised from each animal group (additional recruitment of n=3/treatmentgroup) 1 week after completing the treatment cycles. (FIG. 4H)Kaplan-Meier cumulative survival plot of animals administered withdifferent drugs (n=14/group). The significant differences in survivalbetween the animals treated with i.v. DM1-Bdd and the other groups wasevaluated using the Mantel-Cox log-rank test and the Benjamini Hochbergadjusted p-values. (FIGS. 4I-J) Representative image(s) of bladdersections from the animals in each treatment group (n=3/group). Theorgans were harvested at the end of the 3-week treatment and thenparaffin-embedded, sectioned, and stained with (FIG. 4I) H&E and (FIG.4J) Ki67 (proliferation marker).

FIGS. 5A-H Therapeutic efficacy of DM1-Bdd in treating renal carcinoma.(FIG. 5A) Bright field and fluorescence images of Renca cells that werestably transduced with a lentivirus carrying both GFP and fireflyluciferase genes. Scale bar is 80 µm. (FIG. 5B) Syngeneic xenograftmodel. Representative image of the histological analysis of kidneyscollected from female BALB/c mice (n=3) 1 week after implantation ofmurine Renca cells (4 x 10³ cells/animals) in the renal capsules (blackarrow). Scale bar is 1 mm. (FIG. 5C) Representative mergedbioluminescence/bright field images of animals bearing Renca/GFP-Luctumors after treatment with i.v. PBS (150 µL), i.v. DM1 (0.75 mg/kg, 150µL), i.v. DM1-Bdd (0.75 mg/kg of drug content, 150 µL), or i.t. DM1(0.75 mg/kg, 50 µL) weekly for 3 weeks (n=10/group). (FIG. 5D)Representative photos of the kidneys excised from the animals aftercompletion of the treatment cycle (additional n=4/group). Longitudinalcomparisons of (FIG. 5E) bioluminescence signals at the region ofinterest (ROI = kidney), (FIG. 5F) body weight, and (FIG. 5G) survivalamong animals receiving different treatments (n=14/group). Thesignificant differences in survival between animals treated with i.v.DM1-Bdd and the drugs was evaluated using the Mantel-Cox log-rank testand the Benjamini Hochberg adjusted p-values. (FIG. 5H) Representativekidney sections from animals of each treatment group (additionaln=4/group). The sections were stained with H&E. The green, yellow,black, and blue arrows indicate the presence of pigment-ladenmacrophage, focal mineralization, interstitial fibrosis, and mononuclearcell infiltrates, respectively. Scale bar is 2 mm and 50 µm.

FIGS. 6A-D. DM1-Bdd displays a safe toxicity profile. (FIG. 6A)Representative microscopic images of blood smears collected from femaleBALB/c mice after i.v. administration of PBS, DM1 (0.75 mg/kg), DM1-Bdd(0.75 mg/kg of drug content), or CIS (10 mg/kg) as a positive control,weekly for 3 weeks. Black arrows indicate polychromatophilic macrocytes.Scale bar is 10 µm. (FIG. 6B) Select hematologic results obtained oneweek after completing the different treatment courses. (RBC = red bloodcells and WBC = white blood cells). (Student’s t-test; *p<0.05,**p<0.01, and ***p<0.001). (FIG. 6C) Comparison of select serumbiochemical analytes, including liver enzyme activity (ALP, ALT andAST), muscle enzyme activity (AST and CK), and clearance of nitrogenouswaste (BUN/CREA ratio). (ALP = alkaline phosphatase; ALT = alanineaminotransferase; AST = aspartate aminotransferase; CK = creatinekinase; BUN = blood urea nitrogen; CREA = creatine). (FIG. 6D)Histopathological analysis of the major organs (liver, spleen, heart,lungs, and kidneys) from animals administered with the different drugtreatments. Black arrows indicate the increased of hepatocyte mitoticactivity in liver. Blue arrows show the enhanced hepatic and splenicextramedullary hematopoiesis (EMH). The area in between the white arrowsindicates depletions of erythrocytes and EMH elements in the red pulp ofthe spleen. Red arrows highlight the presence of large and foamymacrophages in the alveoli. Yellow and green arrows indicate flattenedrenal tubular cells and necrotic sloughed debris of dying cellscontained within the lumen, respectively. Scale bar is 30 µm.

FIGS. 7A-B. (FIG. 7A) Table comparing the pharmacokinetic parameters ofthe radiolabeled peptides (n=4/analogue). Blood samples (20 µL) werecollected at various time intervals after tail vein injections of thepeptides or free ⁸⁹Zr (20 µCi, 20 µg, in 100 µL PBS) to the animals. Theresults (radioactivity measured in blood samples) were fit into atwo-compartmental model for determining the PK parameters. (k₁₀ =elimination rate constant; k₁₂ and k₂₁ = transfer rate constant; t_(½α)= half-life of distribution phase; t_(½β) = half-life of eliminationphase; C₀ = threshold of drug concentration for elimination; V_(d) =apparent volume of distribution; CL = total clearance rate; AUC = areaunder the curve; MRT = mean residence time; and V_(SS) = steady statevolume of distribution). (FIG. 7B) End-point biodistribution study of⁸⁹Zr-Bdd. The animals were euthanized at different time intervals(n=4/time point) after administration with the peptide. The amount ofpeptide (radioactivity) in the harvested organs were determined. Theresults (corrected from decay) were expressed as % of injected dose pergram of tissue (%ID/g). (Student’s t-test; *p<0.05, and ***p<0.001).

FIGS. 8A-G. (FIG. 8A) Body weight changes in NSG mice bearing bladdertumors and treated with i.v. PBS (150 µL), i.v. DM1 (0.75 mg/kg, 150µL), i.v. DM1-Bdd (0.75 mg/kg of drug content, 150 µL), i.t. MIT (1mg/mL, 50 µL), i.t. DM1 (0.75 mg/kg, 50 µL), or i.t. DM1-Bdd (0.75mg/kg, 50 µL), weekly for 3 weeks. (FIG. 8B) Comparison of thebioluminescence signal at the region of interest (ROI = bladder) amongthe different drugs. (FIG. 8C) Representative images of bladders excisedfrom 3 animals administered with the different drug treatments. (FIGS.8D-E) Plots comparing the volume (FIG. 8D) and weight (FIG. 8E) of thebladders. (Student’s t-test; *p<0.05, **p<0.01, and ***p<0.001). (FIG.8F) Representative images of bladder sections from animals in eachtreatment group (n=3/group). The organs were harvested at the end of the3-week treatment and then paraffin-embedded, sectioned, and stained withan anti-GFP antibody to identify GFP-expressing tumor cells. (FIG. 8G)Representative images of the bladder sections from the 3 animals whichlacked gross and histologic evidence of tumors after i.v. DM1-Bddtreatment. The animals were sacrificed 210 days after tumor implantationand the organs were harvested, paraffin-embedded, sectioned, and stainedwith H&E, Ki67 (proliferation marker), and an anti-GFP antibody.

FIGS. 9A-E. (FIG. 9A) Representative merged bioluminescence/bright fieldimages of NSG mice bearing orthotopically implanted UMUC-3/GFP-Lucbladder tumors after treatment with i.v. PBS (150 µL), i.v. DOX (5mg/kg, 150 µL), i.v. aldox-Bdd (5 mg/kg of drug content, 150 µL), ori.t. DOX (5 mg/kg, 50 µL), weekly for 3 weeks (n=10/group). Images wereacquired every week to monitor and compare the tumor growth in eachtreatment group. (FIG. 9B) Comparison of the bioluminescence signal atthe region of interest (ROI = bladder) among the different treatmentgroups. (FIG. 9C) Kaplan-Meier cumulative survival plot of animalsadministered the different drugs (n=10/group). The significantdifferences in survival between the animals treated with i.v. aldox-Bddand the other groups was evaluated using the Mantel-Cox log-rank testand the Benjamini Hochberg adjusted p-values. (FIG. 9D) Body weightchanges in NSG mice bearing bladder tumors and treated with PBS, i.v.DOX, i.v. aldox-Bdd, or i.t. DOX weekly for 3 weeks. (FIG. 9E)Representative picture of a mouse treated with i.v. DOX (5 mg/kg) afteradministration of only 2 doses of drug.

FIG. 10 . Extended results of the complete blood count analysis. Tableshowing hematologic results after i.v. administration in BALB/c mice ofPBS, DM1 (0.75 mg/kg), DM1-Bdd (0.75 mg/kg of drug content), or CIS (10mg/kg) used as a positive control, weekly for 3 weeks. (RBC = red bloodcells; HGB = hemoglobin; HCT = hematocrit; MCV = mean corpuscularvolume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscularhemoglobin concentration; RDW = red blood cell distribution width; RET =reticulocyte; PLT = platelet; PDW = platelet distribution width; MPV =mean platelet volume; WBC = white blood cell; NEUT = neutrophil; LYMPH =lymphocyte; MONO = monocyte; EO = eosinophil; and BASO = basophil).

FIG. 11 . Extended results of the serum biochemical analysis. Tableshowing results of all biochemical analytes measured after i.v.administration in BALB/c mice of PBS, DM1 (0.75 mg/kg), DM1-Bdd (0.75mg/kg of drug content), or CIS (10 mg/kg) as a positive control, weeklyfor 3 weeks. (BUN = blood urea nitrogen; CREA = creatinine; ALP =alkaline phosphatase; ALT = alanine aminotransferase; AST = aspartateaminotransferase; GGT = gamma-glutamyl transferase; BIL = bilirubin; TP= total protein; ALB = albumin; GLOB = globulin; A/G = albumin/globulin;P = phosphate; Ca = calcium; GLU = glucose; CHOL = cholesterol; TRIG =triglyceride; CK = creatine kinase; TCO2 = total carbon dioxide; Na =sodium; K = potassium; and CL = chloride).

FIG. 12 . High magnification images of the major organ sections (liver,spleen, lungs, and kidneys) from the animals administered with i.v. DM1(0.75 mg/kg) and i.v. CIS (10 mg/kg) as a positive control, weekly for 3weeks. The sections were stained with H&E. Black arrows indicate theincreased of hepatocyte mitotic activity in liver. Blue arrows showenhanced hepatic and splenic extramedullary hematopoiesis (EMH). Thearea in between white arrows indicates depletions of erythrocytes andEMH elements in the red pulp of the spleen. Red arrows highlight thepresence of large and foamy macrophages in the alveoli. Yellow and greenarrows indicate flattened renal tubular cells and necrotic slougheddebris of dying cells contained within the lumen, respectively. Scalebar is 10 µm.

DETAILED DESCRIPTION OF THE INVENTION

Different approaches have been proposed to improve intravesicalchemotherapy (ITC) treatment. To prolong treatment duration, athermal-sensitive hydrogel, UGN-102, was designed to convert into asemi-solid drug depot inside the bladder and slowly release mitomycin(MIT). A gemcitabine (GEM)-containing semipermeable silicon tube,GemRis, which functions as an osmotic pump, has also been developed tocontrol the release of GEM. Other treatments are currently being testedin clinical in trials for patients who do not respond to BCG.Oportuzumab Monatox is an antibody-protein conjugate that targets tumorcells expressing EpCAM. Adstiladrin is a nonreplicating adenovirusvector that encodes the human IFNα-2b gene. The resulting IFNα-2bproteins, synthesized and expressed in a large quantity, displayed anantitumor activity through inhibition of angiogenesis and induction ofapoptosis in human bladder cancer cells. However, all the aforementionedapproaches are invasive, requiring catherization and/or surgicalprocedures. Alternatively, renal-clearable nanoparticles can be used asdrug carriers. However, they are known to be non-specifically capturedby the reticuloendothelial system, leading to a high off-targetaccumulation in the liver.

Advances in phage display have led to the discovery of many bioactivepeptides that target the urinary system (URS). For example, a galectin-3targeting peptide, G3-C12, has been used for delivering captopril, anangiotensin-converting enzyme inhibitor. Another peptide, (KKEEE)₃K, wasused to carry ciprofloxacin. These peptides were pharmacologicallyactive. They primarily targeted the kidneys via binding to cell-surfacereceptors and have prolonged the post-delivery local retention. Theyhave not been applied for BC treatment. In fact, without chemicalmodifications, a peptide is not a good drug candidate or carrier. Itdisplays unfavorable pharmacokinetics (PK), is rapidly degraded byprotease enzymes, and can be eliminated by renal filtration.

The present invention exploits a peptide’s rapid renal clearance fordisposing treatments to the URS. In certain embodiments, the presentdisclosure provides a small (e.g., 12-amino acid), negatively charged,peptide (e.g., Bdd) that can bypass the recticuloendothelial system andother organs and is preferentially (e.g., exclusively) excreted into theurine with minimal reabsorption. In certain embodiments, the presentdisclosure provides an alternative to ITC, for minimizing off-targetaccumulation in other organs, promoting drug delivery to the URS, andprolonging bladder retention time, to offer a comprehensive and moreeffective treatment of BC (FIG. 1 a ).

The present disclosure relates to a systemically administered peptidedelivery platform that biodistributes to the kidney or urinary tract. Incertain embodiments an active moiety is linked to the peptide deliveryplatform. The disclosure further relates to methods of treating adisease of the kidney or urinary tract in a subject in need thereof. Insome embodiments, the disease is an acute renal disease. In someembodiments, the disease is a chronic renal disease.

In certain embodiments, the peptide delivery platform comprises anegatively charged peptide that can temporary accumulate in kidneys,with no off-target delivery to other organs. Because the peptide iseliminated gradually in urine, it is useful as a drug delivery platformto provide a continuous drug flow for treatment of diseases of thekidney or urinary tract, such as BC. The peptide delivers systemicallyadministered drugs to the urinary system while prolonging bladderretention time, providing a more effective treatment.

In certain aspects, the peptide has multiple negative charges that canpromote rapid renal clearance. Drug delivery to the bladder via renalclearance is thus a continuous event. Compared to drug administrationvia catheterization, the peptide can enhance the therapy’s infiltrationof the entire urinary system with a longer dwell time in the bladder,resulting in a more effective treatment that is non-invasive. In certainaspects, the peptide is used as a drug delivery system for BC treatment,using mertansine (DM1), a highly cytotoxic microtubule inhibitor. DM1 istoo toxic to be used alone but was approved as a pharmacophore inantibody-drug conjugates, such as T-DM1. In certain aspects, the peptideis used to deliver DM1 to treat BC, as well as other cancers of theurinary tract.

In certain aspects, the peptide is used as a drug delivery system fortreatment of a urinary tract infection, for example, with an antibiotic.In certain aspects, the peptide is used as a drug delivery system fortreatment of kidney stones. In certain aspects, the peptide is used as adrug delivery system for treatment of over active bladder. In certainaspects, the peptide is used as a drug delivery system for treatment ofurinary incontinence. In certain aspects, the peptide is used as a drugdelivery system for treatment of interstitial cystitis. In certainaspects, the peptide is used to deliver an imaging agent to the bladder.

In certain aspects, the peptide is water soluble, biologically inert,and non-immunogenic. After IV injection, the peptide can be exclusivelyeliminated via renal clearance with a minimal accumulation in otherorgans, including heart, liver, and spleen.

In certain aspects, the present disclosure provides a compoundrepresented by formula (I):

or a pharmaceutically acceptable salt thereof, wherein the peptide is apeptide targeted for renal clearance.

In certain embodiments, the peptide targeted for renal clearancecomprises a sequence:

wherein one of X, Y and Z is a β-amino acid residue, two of X, Y and Zare independently α-amino acid residues that each have at least one sidechain that comprises a carboxylic acid group, each α-amino acid residuemay independently be of D or L stereochemistry and m is from 2 to 10.

In certain embodiments, the peptide has a zeta potential of from about-30 mV to about +20 mV at physiological pH. In certain embodiments, thepeptide has a zeta potential of from about -20 mV to about 0 mV atphysiological pH. In certain embodiments, the peptide has a zetapotential of from about -5 mV to about 0 mV at physiological pH.

In certain embodiments, the linker comprises one or more groups selectedfrom: amide, imide, thiourea, thioether, disulfide, alkyl, aryl,polyether, hydrazone, ester, carbonate, ketal and silyl ether. Incertain embodiments, the active moiety is a therapeutic agent or animaging agent.

In certain embodiments, the peptide targeted for renal clearancecomprises a sequence:

wherein one of X, Y and Z is a β-amino acid residue two of X, Y and Zare independently α-amino acid residues that each have at least one sidechain that comprises a carboxylic acid group, each α-amino acid residuemay independently be of D or L stereochemistry, m is from 2 to 10, thelinker comprises one or more groups selected from: amide, imide,thiourea, thioether, disulfide, alkyl, aryl, polyether, hydrazone,ester, carbonate, ketal and silyl ether; and the active moiety is atherapeutic agent or an imaging agent.

In certain embodiments, the compound is represented by formula (IA):

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the β-amino acid residue does not comprise anionizable side chain. In certain embodiments, the β-amino acid residueis a β-alanine residue. In certain embodiments, X is a β-alanineresidue. In certain embodiments, each α-amino acid residue isindependently selected from an aspartic acid residue and a glutamic acidresidue. In certain embodiments, at least one α-amino acid residue is anunnatural amino acid residue. In certain embodiments, the unnaturalα-amino acid residue has at least two side chain carboxylic acid groups.In certain embodiments, the unnatural α-amino acid residue is selectedfrom a 2-aminoethane-1,1,2-tricarboxylic acid residue and a2-aminopropane-1,2,3-tricarboxylic acid residue. In certain embodiments,each Y and Z are aspartic acid residues. In certain embodiments, each Yand Z are D-aspartic acid residues. In certain embodiments, X, Y and Zare each independently selected from a β-alanine residue, an asparticacid residue and a glutamic acid residue. In certain embodiments, m is4.

In certain embodiments, the linker comprises a group selected from:

and

In certain embodiments, the linker comprises a group derived fromN-succinimidyl 3-(2-pyridyldithio)propionate or succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

In certain embodiments, the active moiety is a therapeutic agent. Incertain embodiments, the therapeutic agent is selected from ananticancer agent, an antibiotic, an agent that treats overactivebladder, an agent that treats urinary incontinence, an agent that treatsinterstitial cystitis and an agent that treats kidney stones. In certainembodiments, the therapeutic agent is selected from 13-cis-RetinoicAcid, 2-Chlorodeoxyadenosine, 5-Azacitidine. 5-Fluorouracil,6-Mercaptopurine, 6-Thiogua.nine, actinomycin-D, adriamycin,aldesleukin, alemtuzurnab, alitretinoin, all-transretinoic acid, alphainterferon, altretamine, amethopterin, amifostine, anagrelide,anastrozole, arabinosylcylosine, arsenic trioxide, amsacrine,aminocarnptothecin, aminoglutethimide, asparaginase, azacytidine,bacillus calmette-guerin (BCG), bendamustine, bevacizumab, bexarotene,bicalutamide, bortezomib, bleomycin, busulfan, calcium leucovorin,citrovorum factor, capecitahine, canertinib, carboplatin, carmustine,cetuximab, chlorambucil, cisplatin, cladribine, cortisone,cyclophosphamide, cytarabine, darbepoetin alfa, dasatinib, daunomycin,decitabine, denileukin diftitox, dexamethasone, dexasone, dexrazoxane,dactinomycin, daunorubicin, decarbazine, docetaxel, doxorubicin, doxil,aldokonibicirt, doxifluridine, edrecolomab, eniluracil, epirubicin,epoetin alfa, erlotinib, everolimus, exemestane, estramustine,etoposide, filgrastim, fluoxyrnesterone, fulvestranl, flavopiridol,floxuridine, fludarabine, fluorouracil, flutamide, gefitinib,gemcitabine, geiiittizi-ifriab ozogamicin, goserelin, granulocyte-colonystimulating factor, granulocyte macrophage-colony stimulating factor,hexamethylmelamine, hydrocortisone hydroxyurea, ibritumomab, ibritumomabtiuxetan, interferon alpha, interleukin-2, interleukin-11, isotretinoin,ixabepilone, idarubicin, imatinib mesylate, ifosfamide, irinotecan,lapatinib, lenalidomide, letrozole, leucovorin, leuprolide, liposomalAra-C, lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine,mertansine, mesna, methotrexate, methylprednisolone, mitomycin C,mitotane, mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin,oxaliplatin, paclitaxel, pamidronate, pemetrexed, panitumumab, PEGInterferon, pegaspargase, pegfilgrastim, PEG- L-asparaginase,pentosiatin, plicarnycin, prednisolone, prednisone, procarbazine,raloxifene, rituximab, romipicstim, ralitrexed, sapacitabine,sargramostim, satraplatin, sorafenib, sunitinib, semustine,streptozocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus,temozolamide, teniposide, thalidomide, thioguanine, thiotepa, topotecan,toremifene, tositumomab, trastuzumab, trastuzumab emtansine, tretinoin,trimitrexate, alrubicin, vincristine, vinblastine, vindestine,vinorelbine, vorinostat, and zoledronic acid.

In certain embodiments, the therapeutic agent is an anticancer agent. Incertain embodiments, the anticancer agent is selected from mertansine,doxorubicin, dasatinib, cisplatin, mitomycin, gemcitabine andpaclitaxel.

In certain embodiments, X is a β-alanine residue, and Y and Z areD-aspartic acid residues. In certain embodiments, X is a β-alanineresidue, Y and Z are D-aspartic acid residues and m is 4. In certainembodiments, X is a β-alanine residue, Y and Z are D-aspartic acidresidues, m is 4, the linker comprises a disulfide group; and the activemoiety is mertansine.

In certain embodiments, the compound is:

or a pharmaceutically acceptable salt thereof, wherein B is a β-alanineresidue and D is an aspartic acid residue of D or L configuration.

In certain embodiments, the compound is:

or a pharmaceutically acceptable salt thereof, wherein B is a β-alanineresidue and D is an aspartic acid residue of D or L configuration.

In certain embodiments, the present disclosure provides a pharmaceuticalcomposition comprising a compound of the invention. In certainembodiments, the composition is formulated for intravenousadministration.

In certain embodiments, the present disclosure provides a method oftreating cancer, a urinary tract infection, overactive bladder, urinaryincontinence, interstitial cystitis or kidney stones comprisingadministering to a patient in need thereof a compound or composition ofthe invention. In certain embodiments, the present disclosure provides amethod of treating cancer, comprising administering to a patient in needthereof a compound or composition of the invention. In certainembodiments, the cancer is a cancer of the kidney or urinary tract. Incertain embodiments, the cancer is bladder cancer. In certainembodiments, the bladder cancer is non-muscle invasive bladder cancer.In certain embodiments, the bladder cancer is urothelial carcinoma. Incertain embodiments, the compound is administered intravenously.

Pharmaceutical Compositions

The compositions and methods of the present disclosure may be utilizedto treat an individual in need thereof. In certain embodiments, theindividual is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered as a pharmaceutical compositioncomprising, for example, a compound of the disclosure and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known in the art and include, for example, aqueoussolutions such as water or physiologically buffered saline or othersolvents or vehicles such as glycols, glycerol, oils such as olive oil,or injectable organic esters. In preferred embodiments, when suchpharmaceutical compositions are for human administration, particularlyfor invasive routes of administration (i.e., routes, such as injectionor implantation, that circumvent transport or diffusion through anepithelial barrier), the aqueous solution is pyrogen-free, orsubstantially pyrogen-free. The excipients can be chosen, for example,to effect delayed release of an agent or to selectively target one ormore cells, tissues or organs. The pharmaceutical composition can be indosage unit form such as tablet, capsule (including sprinkle capsule andgelatin capsule), granule, lyophile for reconstitution, powder,solution, syrup, suppository, injection or the like. The composition canalso be present in a transdermal delivery system, e.g., a skin patch.The composition can also be present in a solution suitable for topicaladministration, such as a lotion, cream, or ointment.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as acompound of the disclosure. Such physiologically acceptable agentsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. The choice of a pharmaceutically acceptable carrier,including a physiologically acceptable agent, depends, for example, onthe route of administration of the composition. The preparation orpharmaceutical composition can be a self-emulsifying drug deliverysystem or a self-2microemulsifying drug delivery system. Thepharmaceutical composition (preparation) also can be a liposome or otherpolymer matrix, which can have incorporated therein, for example, acompound of the disclosure. Liposomes, for example, which comprisephospholipids or other lipids, are nontoxic, physiologically acceptableand metabolizable carriers that are relatively simple to make andadminister.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

A pharmaceutical composition (or preparation) can be administered to asubject by any of a number of routes of administration including, forexample, orally (for example, drenches as in aqueous or non-aqueoussolutions or suspensions, tablets, capsules (including sprinkle capsulesand gelatin capsules), boluses, powders, granules, pastes forapplication to the tongue); absorption through the oral mucosa (e.g.,sublingually); subcutaneously; transdermally (for example as a patchapplied to the skin); and topically (for example, as a cream, ointmentor spray applied to the skin). The compound may also be formulated forinhalation. In certain embodiments, a compound may be simply dissolvedor suspended in sterile water. Details of appropriate routes ofadministration and compositions suitable for same can be found in, forexample, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231,5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an active compound, such as a compound ofthe disclosure, with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the presentdisclosure with liquid carriers, or finely divided solid carriers, orboth, and then, if necessary, shaping the product.

Formulations of the disclosure suitable for oral administration may bein the form of capsules (including sprinkle capsules and gelatincapsules), cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), lyophile, powders, granules,or as a solution or a suspension in an aqueous or non-aqueous liquid, oras an oil-in-water or water-in-oil liquid emulsion, or as an elixir orsyrup, or as pastilles (using an inert base, such as gelatin andglycerin, or sucrose and acacia) and/or as mouth washes and the like,each containing a predetermined amount of a compound of the presentdisclosure as an active ingredient. Compositions or compounds may alsobe administered as a bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules(including sprinkle capsules and gelatin capsules), tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; (10) complexing agents,such as, modified and unmodified cyclodextrins; and (11) coloringagents. In the case of capsules (including sprinkle capsules and gelatincapsules), tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragees, capsules (including sprinkle capsules andgelatin capsules), pills and granules, may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings well known in the pharmaceutical-formulating art. They may alsobe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms useful for oral administration includepharmaceutically acceptable emulsions, lyophiles for reconstitution,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, cyclodextrins and derivatives thereof, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present disclosure to the body. Suchdosage forms can be made by dissolving or dispersing the active compoundin the proper medium. Absorption enhancers can also be used to increasethe flux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.Pharmaceutical compositions suitable for parenteral administrationcomprise one or more active compounds in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

For use in the methods of this disclosure, active compounds can be givenper se or as a pharmaceutical composition containing, for example, 0.1to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinaceous biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a compound at a particular targetsite.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount may include, but are not limited to, the severityof the patient’s condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with a compound of the disclosure. A larger total dose canbe delivered by multiple administrations of the agent. Methods todetermine efficacy and dosage are known to those skilled in the art(Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the disclosure will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentdisclosure, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans; and other mammals such as equines,cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

In certain embodiments, compounds of the disclosure may be used alone orconjointly administered with another type of therapeutic agent.

The present disclosure includes the use of pharmaceutically acceptablesalts of compounds of the disclosure in the compositions and methods ofthe present disclosure. In certain embodiments, contemplated salts ofthe disclosure include, but are not limited to, alkyl, dialkyl, trialkylor tetra-alkyl ammonium salts. In certain embodiments, contemplatedsalts of the disclosure include, but are not limited to, L-arginine,benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol,diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine,ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium,L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine,potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine,tromethamine, and zinc salts. In certain embodiments, contemplated saltsof the disclosure include, but are not limited to, Na, Ca, K, Mg, Zn orother metal salts. In certain embodiments, contemplated salts of thedisclosure include, but are not limited to, 1-hydroxy-2-naphthoic acid,2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaricacid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid,adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid,benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capricacid (decanoic acid), caproic acid (hexanoic acid), caprylic acid(octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, formic acid, fumaric acid, galactaric acid, gentisic acid,d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid,glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid,hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid,lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid,mandelic acid, methanesulfonic acid , naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid,oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionicacid, 1-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid,succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid acidsalts.

The pharmaceutically acceptable acid addition salts can also exist asvarious solvates, such as with water, methanol, ethanol,dimethylformamide, and the like. Mixtures of such solvates can also beprepared. The source of such solvate can be from the solvent ofcrystallization, inherent in the solvent of preparation orcrystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Definitions

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry, cell and tissue culture,molecular biology, cell and cancer biology, neurobiology,neurochemistry, virology, immunology, microbiology, pharmacology,genetics and protein and nucleic acid chemistry, described herein, arethose well known and commonly used in the art.

The methods and techniques of the present disclosure are generallyperformed, unless otherwise indicated, according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout thisspecification. See, e.g. “Principles of Neural Science”, McGraw-HillMedical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”,Oxford University Press, Inc. (1995); Lodish et al., “Molecular CellBiology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths etal., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co.,N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”,Sinauer Associates, Inc., Sunderland, MA (2000).

Chemistry terms used herein, unless otherwise defined herein, are usedaccording to conventional usage in the art, as exemplified by “TheMcGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill,San Francisco, C.A. (1985).

All of the above, and any other publications, patents and publishedpatent applications referred to in this application are specificallyincorporated by reference herein. In case of conflict, the presentspecification, including its specific definitions, will control.

The term “agent” is used herein to denote a chemical compound (such asan organic or inorganic compound, a mixture of chemical compounds), abiological macromolecule (such as a nucleic acid, an antibody, includingparts thereof as well as humanized, chimeric and human antibodies andmonoclonal antibodies, a protein or portion thereof, e.g., a peptide, alipid, a carbohydrate), or an extract made from biological materialssuch as bacteria, plants, fungi, or animal (particularly mammalian)cells or tissues. Agents include, for example, agents whose structure isknown, and those whose structure is not known. The ability of suchagents to inhibit AR or promote AR degradation may render them suitableas “therapeutic agents” in the methods and compositions of thisdisclosure.

A “patient,” “subject,” or “individual” are used interchangeably andrefer to either a human or a non-human animal. These terms includemammals, such as humans, primates, livestock animals (including bovines,porcines, etc.), companion animals (e.g., canines, felines, etc.) androdents (e.g., mice and rats).

“Treating” a condition or patient refers to taking steps to obtainbeneficial or desired results, including clinical results. As usedherein, and as well understood in the art, “treatment” is an approachfor obtaining beneficial or desired results, including clinical results.Beneficial or desired clinical results can include, but are not limitedto, alleviation or amelioration of one or more symptoms or conditions,diminishment of extent of disease, stabilized (i.e. not worsening) stateof disease, preventing spread of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount.

“Administering” or “administration of” a substance, a compound or anagent to a subject can be carried out using one of a variety of methodsknown to those skilled in the art. For example, a compound or an agentcan be administered, intravenously, arterially, intradermally,intramuscularly, intraperitoneally, subcutaneously, ocularly,sublingually, orally (by ingestion), intranasally (by inhalation),intraspinally, intracerebrally, and transdermally (by absorption, e.g.,through a skin duct). A compound or agent can also appropriately beintroduced by rechargeable or biodegradable polymeric devices or otherdevices, e.g., patches and pumps, or formulations, which provide for theextended, slow or controlled release of the compound or agent.Administering can also be performed, for example, once, a plurality oftimes, and/or over one or more extended periods.

Appropriate methods of administering a substance, a compound or an agentto a subject will also depend, for example, on the age and/or thephysical condition of the subject and the chemical and biologicalproperties of the compound or agent (e.g., solubility, digestibility,bioavailability, stability and toxicity). In some embodiments, acompound or an agent is administered orally, e.g., to a subject byingestion. In some embodiments, the orally administered compound oragent is in an extended release or slow release formulation, oradministered using a device for such slow or extended release.

As used herein, the phrase “conjoint administration” refers to any formof administration of two or more different therapeutic agents such thatthe second agent is administered while the previously administeredtherapeutic agent is still effective in the body (e.g., the two agentsare simultaneously effective in the patient, which may includesynergistic effects of the two agents). For example, the differenttherapeutic compounds can be administered either in the same formulationor in separate formulations, either concomitantly or sequentially. Thus,an individual who receives such treatment can benefit from a combinedeffect of different therapeutic agents.

A “therapeutically effective amount” or a “therapeutically effectivedose” of a drug or agent is an amount of a drug or an agent that, whenadministered to a subject will have the intended therapeutic effect. Thefull therapeutic effect does not necessarily occur by administration ofone dose, and may occur only after administration of a series of doses.Thus, a therapeutically effective amount may be administered in one ormore administrations. The precise effective amount needed for a subjectwill depend upon, for example, the subject’s size, health and age, andthe nature and extent of the condition being treated, such as cancer orMDS. The skilled worker can readily determine the effective amount for agiven situation by routine experimentation.

As used herein, the terms “optional” or “optionally” mean that thesubsequently described event or circumstance may occur or may not occur,and that the description includes instances where the event orcircumstance occurs as well as instances in which it does not. Forexample, “optionally substituted alkyl” refers to the alkyl may besubstituted as well as where the alkyl is not substituted.

It is understood that substituents and substitution patterns on thecompounds of the present disclosure can be selected by one of ordinaryskilled person in the art to result chemically stable compounds whichcan be readily synthesized by techniques known in the art, as well asthose methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

As used herein, the term “optionally substituted” refers to thereplacement of one to six hydrogen radicals in a given structure withthe radical of a specified substituent including, but not limited to:hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl,acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano,haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -OP(O)(O-alkyl)₂ or-CH₂-OP(O)(Oalkyl)₂. Preferably, “optionally substituted” refers to thereplacement of one to four hydrogen radicals in a given structure withthe substituents mentioned above. More preferably, one to three hydrogenradicals are replaced by the substituents as mentioned above. It isunderstood that the substituent can be further substituted.

As used herein, the term “alkyl” refers to saturated aliphatic groups,including but not limited to C₁-C₁₀ straight-chain alkyl groups orC₁-C₁₀ branched-chain alkyl groups. Preferably, the “alkyl” group refersto C₁-C₆ straight-chain alkyl groups or C₁-C₆ branched-chain alkylgroups. Most preferably, the “alkyl” group refers to C₁-C₄straight-chain alkyl groups or C₁-C₄ branched-chain alkyl groups.Examples of “alkyl” include, but are not limited to, methyl, ethyl,1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl,3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl,3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl or 4-octyl and the like.The “alkyl” group may be optionally substituted.

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)-, preferably alkylC(O)-.

The term “acylamino” is art-recognized and refers to an amino groupsubstituted with an acyl group and may be represented, for example, bythe formula hydrocarbylC(O)NH-.

The term “acyloxy” is art-recognized and refers to a group representedby the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkyl” refers to saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-₃₀ for straight chains, C₃₋₃₀ for branchedchains), and more preferably 20 or fewer.

Moreover, the term “alkyl” as used throughout the specification,examples, and claims is intended to include both unsubstituted andsubstituted alkyl groups, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone, including haloalkyl groups such as trifluoromethyland 2,2,2-trifluoroethyl, etc.

The term “C_(x-y)” or “C_(x)-C_(y)”, when used in conjunction with achemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, oralkoxy is meant to include groups that contain from x to y carbons inthe chain. C₀alkyl indicates a hydrogen where the group is in a terminalposition, a bond if internal. A C₁₋₆alkyl group, for example, containsfrom one to six carbon atoms in the chain.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS-.

The term “amide”, as used herein, refers to a group

wherein R⁹ and R¹⁰ each independently represent a hydrogen orhydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom to whichthey are attached complete a heterocycle having from 4 to 8 atoms in thering structure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

or

wherein R⁹, R¹⁰, and R¹⁰’ each independently represent a hydrogen or ahydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom to whichthey are attached complete a heterocycle having from 4 to 8 atoms in thering structure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “carbamate” is art-recognized and refers to a group

or

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbylgroup.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbocycle” includes 5-7 membered monocyclic and 8-12 memberedbicyclic rings. Each ring of a bicyclic carbocycle may be selected fromsaturated, unsaturated and aromatic rings. Carbocycle includes bicyclicmolecules in which one, two or three or more atoms are shared betweenthe two rings. The term “fused carbocycle” refers to a bicycliccarbocycle in which each of the rings shares two adjacent atoms with theother ring. Each ring of a fused carbocycle may be selected fromsaturated, unsaturated and aromatic rings. In an exemplary embodiment,an aromatic ring, e.g., phenyl, may be fused to a saturated orunsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Anycombination of saturated, unsaturated and aromatic bicyclic rings, asvalence permits, is included in the definition of carbocyclic. Exemplary“carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane,1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene,bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fusedcarbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene,bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene andbicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one ormore positions capable of bearing a hydrogen atom.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbonate” is art-recognized and refers to a group —OCO₂—.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The term “ester”, as used herein, refers to a group —C(O)OR⁹ wherein R⁹represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heterocyclyl” and “heterocyclic” also include polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a =O or =S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are consideredto be hydrocarbyl for the purposes of this application, but substituentssuch as acetyl (which has a =O substituent on the linking carbon) andethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbylgroups include, but are not limited to aryl, heteroaryl, carbocycle,heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer atoms in the substituent,preferably six or fewer. A “lower alkyl”, for example, refers to analkyl group that contains ten or fewer carbon atoms, preferably six orfewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl,or alkoxy substituents defined herein are respectively lower acyl, loweracyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy,whether they appear alone or in combination with other substituents,such as in the recitations hydroxyalkyl and aralkyl (in which case, forexample, the atoms within the aryl group are not counted when countingthe carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 5 to 7.

The term “sulfate” is art-recognized and refers to the group —OSO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the grouprepresented by the general formulae

or

wherein R⁹ and R¹⁰ independently represents hydrogen or hydrocarbyl.

The term “sulfoxide” is art-recognized and refers to the group—S(O)—.

The term “sulfonate” is art-recognized and refers to the group SO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group —S(O)₂—.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group.

The term “thioester”, as used herein, refers to a group —C(O)SR⁹ or-SC(O)R⁹ wherein R⁹ represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

The term “urea” is art-recognized and may be represented by the generalformula

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbyl.

The term “modulate” as used herein includes the inhibition orsuppression of a function or activity (such as cell proliferation) aswell as the enhancement of a function or activity.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, excipients, adjuvants,polymers and other materials and/or dosage forms which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable salt” or “salt” is used herein to refer toan acid addition salt or a basic addition salt which is suitable for orcompatible with the treatment of patients.

The term “pharmaceutically acceptable acid addition salt” as used hereinmeans any non-toxic organic or inorganic salt of any base compoundsrepresented by Formula I. Illustrative inorganic acids which formsuitable salts include hydrochloric, hydrobromic, sulfuric andphosphoric acids, as well as metal salts such as sodium monohydrogenorthophosphate and potassium hydrogen sulfate. Illustrative organicacids that form suitable salts include mono-, di-, and tricarboxylicacids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric,fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic,phenylacetic, cinnamic and salicylic acids, as well as sulfonic acidssuch as p-toluene sulfonic and methanesulfonic acids. Either the mono ordi-acid salts can be formed, and such salts may exist in either ahydrated, solvated or substantially anhydrous form. In general, the acidaddition salts of compounds of Formula I are more soluble in water andvarious hydrophilic organic solvents, and generally demonstrate highermelting points in comparison to their free base forms. The selection ofthe appropriate salt will be known to one skilled in the art. Othernon-pharmaceutically acceptable salts, e.g., oxalates, may be used, forexample, in the isolation of compounds of Formula I for laboratory use,or for subsequent conversion to a pharmaceutically acceptable acidaddition salt.

The term “pharmaceutically acceptable basic addition salt” as usedherein means any non-toxic organic or inorganic base addition salt ofany acid compounds represented by Formula I or any of theirintermediates. Illustrative inorganic bases which form suitable saltsinclude lithium, sodium, potassium, calcium, magnesium, or bariumhydroxide. Illustrative organic bases which form suitable salts includealiphatic, alicyclic, or aromatic organic amines such as methylamine,trimethylamine and picoline or ammonia. The selection of the appropriatesalt will be known to a person skilled in the art.

Many of the compounds useful in the methods and compositions of thisdisclosure have at least one stereogenic center in their structure. Thisstereogenic center may be present in a R or a S configuration, said Rand S notation is used in correspondence with the rules described inPure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates allstereoisomeric forms such as enantiomeric and diastereoisomeric forms ofthe compounds, salts, prodrugs or mixtures thereof (including allpossible mixtures of stereoisomers). See, e.g., WO 01/062726.

Furthermore, certain compounds which contain alkenyl groups may exist asZ (zusammen) or E (entgegen) isomers. In each instance, the disclosureincludes both mixture and separate individual isomers.

Some of the compounds may also exist in tautomeric forms. Such forms,although not explicitly indicated in the formulae described herein, areintended to be included within the scope of the present disclosure.

“Prodrug” or “pharmaceutically acceptable prodrug” refers to a compoundthat is metabolized, for example hydrolyzed or oxidized, in the hostafter administration to form the compound of the present disclosure(e.g., compounds of formula I). Typical examples of prodrugs includecompounds that have biologically labile or cleavable (protecting) groupson a functional moiety of the active compound. Prodrugs includecompounds that can be oxidized, reduced, aminated, deaminated,hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated,dealkylated, acylated, deacylated, phosphorylated, or dephosphorylatedto produce the active compound. Examples of prodrugs using ester orphosphoramidate as biologically labile or cleavable (protecting) groupsare disclosed in U.S. Pat. 6,875,751, 7,585,851, and 7,964,580, thedisclosures of which are incorporated herein by reference. The prodrugsof this disclosure are metabolized to produce a compound of Formula I.The present disclosure includes within its scope, prodrugs of thecompounds described herein. Conventional procedures for the selectionand preparation of suitable prodrugs are described, for example, in“Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filter, diluent, excipient, solvent or encapsulatingmaterial useful for formulating a drug for medicinal or therapeutic use.

The term “Log of solubility”, “LogS” or “logS” as used herein is used inthe art to quantify the aqueous solubility of a compound. The aqueoussolubility of a compound significantly affects its absorption anddistribution characteristics. A low solubility often goes along with apoor absorption. LogS value is a unit stripped logarithm (base 10) ofthe solubility measured in mol/liter.

EXAMPLES

The disclosure now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present disclosure, and are not intended to limit the disclosure.

Materials and Methods

Chemicals and Supplies -2(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate(HBTU) and the N-hydroxybenzotriazole (HOBt) were purchased fromVivitide (Gardner, MA). All protected amino acids, rink amide MBHAresin, and N-methylmorpholine (NMM) were supplied by Gyros ProteinTechnologies (Tucson, AZ). Polyethylene glycol (PEG₃) was obtained fromCreative PEGWorks (Durham, NC). Trifluoroacetic acid (TFA), piperidine,thioanisole, anisole, 1,2-ethanedithiole, methyl-tert-butyl ether,N,N-diisopropylethylamine (DIPEA), dimethylformamide (DMF), acetonitrile(ACN), N-methyl-2-pyrrolidone (NMP), sodium carbonate, Sephadex G25,L-glutathione reduced (GSH), lipopolysaccharide (LPS), andcis-dichlorodiammine platinum (II) (cisplatin) were purchased from SigmaAldrich (Saint-Louis, MO). Succinimidyl 3-(2-pyridyldithio)propionate(SPDP) was supplied by Invitrogen (Carlsbad, CA). p-SNC-deferoxamine(DFO) was from Macrocyclics Inc. (Plato, TX).N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine (DM1) andaldoxorubicin HC1 (aldox) were from MedKoo Biosciences (Morrisville,NC). Doxorubicin (DOX) and gemcitabine (GEM) were obtained from LCLaboratories (Woburn, MA). Mitomycin (MIT) was supplied by Selleckchem(Houston, TX). Cyanine5.5 NHS ester was purchased from LumiprobeCorporation (Hallandale Beach, FL) and luciferin was from CaliperLifeScience (Hopkinton, MA).

Peptide synthesis - All peptides were synthesized on a solid-phasepeptide synthesizer (PS3, Gyros Protein Technologies, Tucson, AZ) usingthe N-α-Fmoc methodology on Rink amide resin, as previously described.The side-chain protected amino acids (0.4 mmol) were attached to theresin (385 mg, 0.1 mmol) by stepwise elongation using NMM as a base,HBTU/HOBt as coupling reagents, and piperidine (20% in DMF v/v) as adeprotecting agent. A β-alanine was incorporated at the peptideN-terminal as a spacer for further conjugation of the desired moieties,including fluorophore (Cyanine5.5), chelator (DFO), or linker (SPDP)prior to peptide cleavage.

Immunogenicity assay - Female BALB/c mice were treated with a singledose of Bdd or LPS (5 mg/kg) via tail-vein injection (n=3/group). Miceadministered with PBS were used as a negative control. Blood sampleswere collected 4 h later via retro-orbital sinus puncture technique, andthe concentration of the innate immune and inflammatory cytokines IL1β,IL2, IL6, IL10, TNF-α, and INF-γ in plasma was measured with commercialsandwich enzyme-linked immunosorbent assay kits. ELISA assays wereperformed according to the manufacturer’s instructions (Invitrogen,Carlsbad, CA).

Methods to incorporate different functionalities to peptide - Cyanine5.5NHS ester (50 mg, 1.4 eq) in DMF (4 mL), DFO (25 mg, 1 eq) in DMSO (4mL), or SPDP (25 mg, 1 eq) in NMP (4 mL), were added to the resin (0.05mmoL, 1 eq) and allowed to react overnight at room temperature. Forfluorophore and chelator conjugation, the reactions were performed inpresence of an organic base (DIPEA, 1 mL). The peptides were thenremoved from the resin using a cleaving cocktail (5 mL) containingTFA/thioanisole/1,2-ethandithiol /anisole (90:5:3:2) for 4 h andprecipitated in methyl-tert-butyl ether. The resulting peptides(Cy-peptide, DFO-peptide, and SPDP-peptide) were purified to >98% purityusing reverse-phase high performance liquid chromatography (rp-HPLC,Agilent, Santa Clara, CA) and were characterized by MALDI-TOF analysis(Tufts Medical School, Boston, MA) to confirm their molecular weights.

Synthesis of cleavable drug-peptide conjugate - DM1 (1 mg, 1 eq) wasadded to SPDP-peptide (10 mg, 3 eq) in a cosolvent of NMP (100 µL) andphosphate-buffered saline (PBS; 10 mM, pH 7, 100 µL), and allowed toreact for 2 days at room temperature. The DM1-peptide was then purifiedby rp-HPLC. To conjugate aldox, a thiol-reactive side (cysteine) wasintroduced at the peptide N-terminal. Aldox (2 mg, 1 eq) was added tothe peptide (10 mg, 3 eq) in PBS (10 nM, 1 mL, pH 7.4), and allowed toreact for 30 minutes. The drug-peptide obtained was pH-sensitive andthereby was purified by rp-HPLC in neutral conditions (mobile phase A:PBS, mobile phase B: 90% ACN in PBS). Size exclusion chromatography(TipTop C-18 column) was used to remove the salt content. All the finaldrug-peptide conjugates were characterized using MALDI-TOF analysis andwere quantified with UV absorbance, according to the pre-determinedextinction coefficient of DM1 (ε = 3,700 cm⁻¹ M⁻¹) or aldox (ε =13,000cm⁻¹ M⁻¹) in 5% (v/v) PBS in methanol.

Radiochemistry - ⁸⁹Zirconium (⁸⁹Zr) was supplied by 3DImaging LLC(Little Rock, AR). The ⁸⁹Zr-oxalate (500 µCi) was first neutralized withan equivalent volume of sodium carbonate solution (2 M), and then addedto the DFO-peptides (0.2 mg, 250 µL). After incubation for 1.5 h at roomtemperature, the radiolabeled peptides (⁸⁹Zr-peptides) were purified bysize exclusion chromatography using Sephadex G-25 gel to remove the freezirconium.

Pharmacokinetic study - BALB/c mice (Jackson Laboratory, Bar Harbor, ME)were administered with ⁸⁹Zr-peptide or free ⁸⁹Zr (20 µCi, 100 µL) viatail-vein injections (n=4/condition). Blood samples (20 µL) werecollected at various time intervals, using retro-orbital sinus puncturetechnique. The radioactivities were measured on Wallac Wizard 2 gammacounter (Perkin-Elmer, Waltham, MA). The pharmacokinetic models andparameters of ⁸⁹Zr-peptides and free ⁸⁹Zr, including the serum half-lifeand the plasma clearance, were estimated by fitting the data forcompartmental model selection using PKSolver 2.0 software.

µPET/CT imaging and biodistribution study - BALB/c mice wereintravenously injected with different ⁸⁹Zr-peptide analogues (20 µCi,100 µL). µPET/CT imaging was performed in animals with and without urinecollection prior to the first imaging (n=4/analogue/condition).Whole-body images were acquired 1, 4, and 24 h after injection, usingInveon µPET/CT scanner (Siemens Medical Solutions, Malvern, PA). µPET/CTmaximum energy projections were processed using Amide v1.0.4 and InveonResearch Workplace software. The radioactivities at different region ofinterest (ROI) were determined. For end-point biodistribution studies,the mice were euthanized 0.17, 1, 2, 5, and 7 days after treatment with⁸⁹Zr-peptides (n=3/peptide analogue/time point). The radioactivities ofthe harvested organs were measured using the Wallac Wizard 2 gammacounter. The results were corrected from radioactive decay, and wereexpressed as a percentage of the injected dose (%ID) or percentage ofinjected dose per gram of tissue (% ID/g).

Fluorescence imaging - Female SHO mice (Charles River Laboratories,Wilmington, MA) were administered with free Cyanine5.5 or Cy-peptideanalogues (0.5 nmol of Cyanine5.5 content measured by UV absorbance at680 nm) in a PBS (150 µL) via tail-vein injections (n=4/group).Real-time fluorescence imaging was performed using In vivo Xtremeimaging system (Bruker, Billerica, MA). Whole body fluorescence imageswere acquired 1 and 4 h post injection using the appropriate excitation(670 nm) and emission (750 nm) filters. The animals were theneuthanized. The organs were excised to perform ex vivo fluorescenceimaging. Imaging was also performed on urine samples (50 µL) collectedfrom separate animals 1 h after i.v. injection of free dye or Cy-peptideanalogues (n=4/treatment). Bruker MI software was used to process thefluorescence/bright light images and measure the fluorescence intensityin different ROIs. All the data were corrected to eliminate the organ orfluid auto-fluorescence.

Cell lines - MB49 was supplied by EMD Millipore Corporation (Temecula,CA). UMUC-3, T24, and Renca were obtained from ATCC (Manassas, VA). Eachcell line was cultured according to the company’s instructions.Mycoplasma tests (Lonza, Basel, Switzerland) were performed periodicallyto ensure that there was no contamination. Both UMUC-3 and Renca celllines were further transduced with GlowCell 16 FLuc-F2A-GFP lentivirus(Biosettia, San Diego, CA) carrying both firefly luciferase and greenfluorescent protein (GFP) genes. Briefly, the cells were seeded in6-well plates (0.25 x 10⁶ cells/well) and incubated for 3 days with thevirus (2x10⁷ IU/well) in the presence of polybrene (10 µg/mL). To ensuremore than 95% of cell purity, the cell lines were analyzed and sortedfor GFP expression using flow cytometry. The successful transduction wasfurther confirmed by imaging, using EVOS FL Auto Fluorescence microscope(Life Technology, Carlsbad, CA).

Cell viability and cytotoxicity assay - Cancer cells (3 x 10³/well) wereseeded on a flat bottom 96-well plate overnight. Differentconcentrations of DM1, gemcitabine (GEM), mitomycin (MIT), cisplatin(CIS), doxorubicin (DOX), aldoxorubicin (aldox), or drug-loaded peptides(DM1-peptide and aldox-peptide), were then added to the cells for 72 hand then washed 2 times with PBS (400 µL). CellTiter Glo reagent(Promega, Madison, WI) was added in each well (50 µL). The luminescencegenerated was recorded using a microplate reader (Tecan US Inc.,Morrisville, NC). The dose response curves were plotted, and the halfmaximal inhibitory concentrations (IC₅₀ values) were calculated usingGraph Pad Prism 6.0 software.

In vitro drug release study - DM1-peptide conjugates (10 µM of drugcontent) were incubated in the presence of the reducing agent GSH (1 mM)in PBS buffer (800 µL). At different time intervals (0, 2, 4, 6, 8, 12,24, and 48 h), a small amount of solution (100 µL) was taken out forHPLC analysis using C18 analytical column. The amount of DM1 activemetabolites released over time was measured (absorbance detected at 254nm) and then quantified. The experiment was performed independently intriplicate. A similar experiment was performed to quantify the releaseof aldox. Aldox-peptide conjugates (100 µM of drug content) wereincubated in PBS buffer (800 µL) with different pH (7.4 and 5.5) in aglass vial coated with silica to avoid non-specific adsorption on thereaction vessel surfaces. The amount of drug released over time was thenmeasured as described above (absorbance detected at 480 nm). Foraccurate quantification, all the results were normalized compared to aDOX control incubated in similar conditions.

Animal care - All animals used for this project were housed in apathogen-free barrier room, maintained at a controlled temperature (72 ±2° F.) with daily 12 h cycle of light and dark. All the proceduresconducted on mice were approved by the Weill Cornell Medical CenterInstitutional Animal Care and Use Committee (protocol # 2019-0003), andwere consistent with the recommendations of the American VeterinaryMedical Association and the National Institutes of Health Guide for theCare and Use of Laboratory Animals. The mice were allowed to acclimatefor at least 7 days before performing any experiment. ImmunocompromisedNSG mice are susceptible to infection. Thus, the animals were fed withdiet containing sulfatrim antibiotic (Envigo, Indianapolis, IN). Animalsused for fluorescence imaging were fed with AING93 non-fluorescence diet(Envigo, Indianapolis, IN).

Therapeutic efficacy of drug-Bdd conjugates for treating BC - Animalswere orthotopically implanted with tumors as previously described.Briefly, the urine of 7- to 9-week-old female NSG mice (JacksonLaboratory, Bar Harbor, ME) was remove through a sterile 24 G pediatricvenous catheter (Dublin, Ireland). A trypsin solution (0.125%, 80 µL)was then delivered into the bladder. Subsequently, UMUC-3/GFP-Luc cells(4 x 10⁴ cells) in culture media (50 µL) were transferred into thebladders and allowed for seeding. Tumor progression was monitored bybioluminescence imaging. Luciferin (3 mg) in PBS (100 µL) was given tothe animals (via intraperitoneal injection) 15 min prior to performingimaging. Once tumor progression was confirmed in bladders (based onbioluminescence signal), the animals were randomly assigned for weeklytreatment with PBS, DM1, or DM1-Bdd (0.75 mg/kg of drug content) insaline (150 µL) via tail-vein injection for 3 weeks (n=14/group).Separate groups of animals were assigned for intravesical DM1, DM1-Bdd(0.75 mg/kg of drug content), or MIT (1 mg/mL) in saline (50 µL)treatments (n=14/group). The therapeutic efficacy was evaluated based ontumor growth inhibition (bioluminescence imaging) and long-termsurvival. Additional mice were recruited for histopathological analysis(n=4/group). These animals were immediately euthanized 1 week after thecompletion of the treatment schedule. The bladders were harvested andpreserved in neutral buffered formalin (10%). The same experimentalconditions were used for comparing the treatment outcomes among animalstreated with PBS, aldox, or aldox-Bdd via tail-vein injection orintravesical administration (5 mg/kg of drug content).

Evaluation of DM1-Bdd for Kidney cancer treatment - Renca/GFP-Luc cells(4 x 10³ cells) in PBS (3 µL) were orthotopically implanted into thekidney capsules of 7- to 9-week-old female BALB/c mice (JacksonLaboratory, Bar Harbor, ME) as previously described. The tumorprogression was confirmed by bioluminescence imaging. The animals wererandomly assigned into 4 groups for weekly treatment with PBS, DM1, orDM1-Bdd (0.75 mg/kg of drug content) through tail-vein injection, orDM1-Bdd (0.75 mg/kg of drug content) through intravesical administration(n=14/treatment). The animals were monitored for tumor growth inhibitionand survival as described above. Additional mice were used forhistopathological analysis (n=4/group).

Histologic analysis - The tissue samples were fixed in formalin,dehydrated with ethanol, and embedded in paraffin. The tissue sections(5 µm) were stained with hematoxylin and eosin Y (H&E). Forimmunohistochemistry, the bladder sections were deparaffined and thenrehydrated before incubation with anti-Ki67 or anti-GFP antibodies(Abcam, Cambridge, UK) overnight. Slides were then counterstained withHematoxylin. High resolution images were acquired using Aperio 9 DigitalPathology slide scanner (Leica Biosystems, Weltzar, Germany).

Necropsy - Seven- to 9-week-old female BALB/c mice (n=4/group) weretreated weekly with PBS, DM1, DM1-peptide (0.75 mg/kg of drug content),or cisplatin (10 mg/kg) via tail-vein injection (150 µL). At the end ofa 3-week treatment, mice were euthanized. The organs/tissues wereharvested, fixed in 10% neutral buffered formalin for 2 days. Bones weredecalcified in a formic acid solution (Surgipath Decalcifier I; LeicaBiosystems, Weltzar, Germany). The samples were then embedded inparaffin, sectioned (5 µm), and stained with H&E for examination by anACVP board certified anatomic pathologist.

Nephrotoxicity studies - PBS, DM1, DM1-peptide, MIT, GEM, CIS (0.75mg/kg of drug content), or a high dose of CIS (10 mg/kg), wereadministered to BALB/c mice via the tail veins (n=3/group). The level ofthe acute renal injury biomarkers, neutrophil gelatinase-associatedlipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in the urinesamples, were measured using ELISA assays according to themanufacturer’s instructions (R&D Systems, Minneapolis, MN). Thecomplementary histopathological analyses were performed on the kidneysof animals treated with PBS, DM1, DM1-Bdd, (0.75 mg/kg of drug content),or a high dose of CIS (10 mg/kg). All the animals (n=3/group) wereeuthanized at the end of the treatment and the kidneys were collectedand fixed in 10% neutral buffered formalin. The tissues were thenprocessed, embedded in paraffin, sectioned, and stained with H&E.Immunohistochemistry (IHC) for kidney injury (NGAL and KIM-1) was alsoperformed.

Hematology and biochemistry - Blood samples were collected via cardiacpuncture. A complete blood count, including red blood cell, white bloodcell, reticulocyte and platelet counts, with an automated differentialleukocyte count, was performed using an IDEXX Procyte DX hematologyanalyzer (iDEXX, Westbrook, ME). Blood smears were prepared, stainedwith modified Wright’s stain, examined in a blinded fashion by aboard-certified veterinary clinical pathologist (T.S.), and imaged usinga Nikon Eclipse TE2000-U fluorescent microscope equipped with aphotometrics CoolSNAP HQ² camera (Nikon Corporation, Tokyo, Japan). Theblood was also centrifuged (1,500xG) for 15 min to obtain the serum forbiochemical analysis using a Beckman Coulter AU680 analyzer (BeckmanCoulter, Brea, CA).

Statistical analysis - Statistical analyses were performed using GraphPad Prism 7.0 software and R v4.0.5 (R Foundation for StatisticalComputing, Vienna, Austria) software. All data are presented as mean ±standard deviation and significances were assigned at *p<0.05, **p<0.01,and ***p<0.001. Significant differences between groups were determinedusing a 2-tailed Student’s t-test. For survival evaluation, theMantel-Cox Log-Rank test were performed to compare the survival curvesof animals treated with i.v. DM1-Bdd or aldox-Bdd to other treatments.P-values were adjusted for multiple comparisons using the Benjamini &Hochberg method. All p-values are two-sided with statisticalsignificance evaluated at the 0.05 alpha level. Ninety-five percent(exact) confidence intervals for all parameters were calculated toassess the precision of the obtained estimates.

Example 1: Bdd Can Be Exclusively Eliminated via Renal Clearance

Bdd was designed in D-configuration to avoid degradation by proteaseenzymes in the blood circulation. It is composed of multiple D-asparticacid (d) and (β-alanine (B) residues (FIG. 1 b ). The aspartic acidscontributed to the overall negative charge, preventing non-specificuptake by major organs and promoting renal clearance of the peptide. TheB residues served as linkers to avoid the formation of a secondarystructure. To investigate how the charges impacted Bdd’s in vivobehavior, a panel of Bdd analogues were synthesized that were inL-configuration (BDD), neutral (BKD), carrying positive charges (BKK),and in pegylated form (PEG₃DD) for comparative studies. The peptideswere labeled with 89-zircronium (⁸⁹Zr), a long-lived radioisotope(t_(½)=78 h), which allowed the study of the pharmacokinetics (PK) andlong-term biodistribution (BD) using micro positron emission andcomputerized tomography (µPET/CT) imaging. The radiolabeled peptides(⁸⁹Zr-peptides) were synthesized by firstly conjugating a deferoxamine(DFO) chelator to the peptide in solid phase after amino acidelongation. ⁸⁹Zr was then complexed with the resulting DFO-peptideconjugates in solution under a basic conditions (FIG. 1 c ).

The results showed that ⁸⁹Zr-Bdd and ⁸⁹Zr-BDD displayed minimaloff-target delivery (FIG. 1 d ). Radioactivity was not detected in themajor organs, except the kidneys. Both peptides were rapidly excretedinto the urine within 1 h after i.v. administration, as confirmed by theabsence of radioactivity in the bladders that were catheterized andemptied prior to imaging. The results also showed that as high as 80% ofthe total injected dose (ID) was in the first urine sample collectedfrom the animals injected with ⁸⁹Zr-Bdd (FIG. 1 e ), suggesting that thepeptide was filtered via the glomerulus with minimal reabsorption. Onthe other hand, the positively charged ⁸⁹Zr-BKK was delivered to theliver in addition to the URS (FIG. 1 d ). Renal uptake among the peptideanalogues was also compared. Except for an initial increase in PEG₃DD,they all showed reduced accumulation in kidneys over time (FIG. 1 f ).To determine the PK profile, experimental data was fit into atwo-compartment model. The results showed that all the peptide analoguesdisplayed shorter half-lives compared to the free ⁸⁹Zr (FIG. 1 g ).⁸⁹Zr-Bdd had a terminal half-life of 0.53 h and rapid plasma clearance(FIG. 7 a ). An end-point biodistribution study of ⁸⁹Zr-Bdd was thenperformed. The results were in good agreement with the imaging and PKstudies. No peptide in major organs or blood circulation was detected 4h after i.v. injection (FIG. 1 h & FIG. 7 b ). Despite of the rapidclearance, a 15.1% of ⁸⁹Zr-Bdd’s ID was found in kidneys, whichdecreased to 2.1% after 7 days.

Example 2: As a Carrier of Hydrophobic Molecule

Most chemotherapeutics are hydrophobic molecules that displayunfavorable PK and BD, leading to off-target delivery and undesiredtoxicity. Bdd’s rapid renal clearance could be beneficial for promotingdrug delivery to the URS. To demonstrate that, hydrophobic Cyanine5.5fluorophore (Cy) was covalently attached as a drug model to the peptideanalogues (FIG. 2 a ) and compared the resulting conjugates (Cy-peptide)for in vivo delivery. Both Cy-Bdd and Cy-BDD could be rapidly eliminatedvia renal clearance. They reached the animals’ bladders 1 h after i.v.injections (FIG. 2 b ), with as high as 70-75% ID presented in the urine(FIG. 2 c ). On the other hand, Cy-BKD, Cy-BKK, Cy-PEG₃DD, and free Cywere mainly taken up by the liver. Ex vivo imaging of the harvestedorgans (FIG. 2 d ) was also performed. As expected, the accumulation ofCy-Bdd and Cy-BDD in kidneys was minimal compared to other conjugates(FIG. 2 e ). However, unlike ⁸⁹Zr-Bdd, trace amounts of Cy-Bdd andCy-BDD were taken up by the stomach, liver, and intestines. Thebiodistribution differences were likely attributed to the replacement ofa negatively charged ⁸⁹Zr-DFO by the hydrophobic Cy. Overall, Bdd couldbe used for delivering hydrophobic molecules, such as Cy, to the URSwhile maintaining the comprehensive UDD properties.

Example 3: As Carrier of Chemotherapeutics

The Bdd’s excellent UDD properties prompted further investigation ofdelivery of chemotherapeutics for NMIBC treatment. Many peptides areimmunogenic. It was first confirmed that the Bdd peptide did not triggerinnate immune responses. There was no increase of inflammatory cytokinelevels (IL-1β, IL-2, IL-6, IL-10, TNF-α, and INF-γ) in the plasma ofBALB/c mice 4 h after i.v. injection (FIG. 3 a ). Using the fluorescenceCy-Bdd, it was confirmed that the peptide could be taken up by human BC(UMUC-3) and murine kidney cancer (Renca) cells (FIG. 3 b ). Overlappingof the peptide (red) and lysosome (green) fluorescence was observed,suggesting that cellular uptake of the peptide mainly occurred viaendocytosis. As proof-of-principle, emtansine (DM1) was selected, apotent microtubule inhibitor, as a drug candidate. Compared to otherconventional chemotherapeutics, DM1 was more potent than cisplatin(CIS), doxorubicin (DOX) and mitomycin (MIT), and as effective asgemcitabine (GEM) against UMUC-3, with IC₅₀ values at the nanomolar (nM)range (FIG. 3 c ). The drug was also effective against Renca cell line.Next, a DM1-Bdd conjugate was synthesized by attaching DM1 to Bdd via acleavable disulfide linker (FIG. 3 d ), enabling drug release in areducing environment, such as in the presence of intracellularglutathione (GSH) (FIG. 3 e ). The chemistry for conjugating otherchemotherapeutics was also established, such as aldoxorubicin (aldox),to Bdd (FIG. 3 f ). Aldox is a DOX derivative modified with a hydrazonelinker, which is known to be sensitive to the acidic lysosome and tumormicroenvironment. The drug release from aldox-Bdd conjugate waspH-dependent (FIG. 3 g ). In terms of cytotoxicity, both DM1-Bdd andaldox-Bdd exhibited a similar potency to the corresponding free drugsagainst different murine (MB49) and human (UMUC-3 and T24) BC cell lines(FIGS. 3 h-i ).

Example 4: A More Effective Alternative to Conventional ITC

Unlike with intravesical (i.t.) administration, animals did not need tovoid shortly after i.v. injection (FIG. 4 a ). DM1-Bdd, administeredthrough i.v. injection, should prolong the drug’s bladder-dwelling time.This, together with the unique UDD properties, should offer a moreeffective (compared to ITC) and safer (compared to systemicchemotherapy) therapeutic option when using Bdd as a drug carrier.However, Bdd could temporarily accumulate in kidneys (FIG. 1 d and h).Therefore, prior to evaluating the therapeutic efficacy of DM1-Bdd, thetolerability of kidney to a single injectable dose in healthy mice wasassessed. The results showed that DM1-Bdd slightly increased the levelsof urinary tubular injury markers, kidney injury molecule-1 (KIM-1) andneutrophil gelatinase-associated lipocalin (NGAL), in the urine 1 dayafter the injection (FIG. 4 b ). The increases were minimal andtransient, returning to basal levels within 3 days. Histologicexamination at 3 days after drug administration did not reveal anyabnormalities in the kidneys, with no elevation of the immunoreactivityof the tissue sections for KIM-1 and NGAL, when compared to PBS (FIG. 4c ). In contrast, other i.v. chemotherapeutics, including DM1, MIT, CIS,and GEM, showed a continued induction of KIM-1 and/or NGAL at this timepoint. Dilation and degeneration of tubular epithelium in animalstreated with a high dose of i.v. CIS (positive control) was also found(FIG. 4 c ).

DM1-Bdd for treating mice bearing orthotopic human UMUC-3 tumors wasthen evaluated. Prior to implanting UMUC-3 cells into the animals’bladders, they were stably transduced with a dual luciferase and GFPreporter (FIG. 4 d ). This allowed monitoring of the disease progressionwith bioluminescence imaging. It was also confirmed that the establishedtumors, growing in the lamina propia, were non-invasive in nature andlimited to the urinary bladder submucosa (FIG. 4 e ). Compared to theclinically used ITC (i.t. MIT), both i.v. DM1 and DM1-Bdd were moreeffective in inhibiting tumor growth (FIGS. 4 f-g and FIGS. 8 a-e ) andprolonged animal survival (FIG. 4 h ). An i.v. injection of DM1-Bddprovided a better treatment outcome when compared to i.t.administration. According to the imaging data acquired during the 3once-per-week treatments course, the anti-tumor activity of i.v. DM1-Bddand DM1 was similar (FIG. 4 f ). However, those animals treated withi.v. DM1-Bdd showed a significant improvement in overall survival (36%versus 0% survived after 100 days). In a separate experiment, thetherapeutic effect on tumor reduction was confirmed by histology. Tumorsof the animals treated with i.v. DM1-Bdd were smaller (FIG. 4 i ).Immunohistochemistry revealed fewer GFP-positive cells and lowerproportion of cells expressing the proliferation marker (Ki67),confirming the inhibition of tumor cell growth and proliferation (FIG. 4j and FIG. 8 f ). More importantly, DM1-Bdd treatment was curative for21% of the animals. The survivor mice lacked gross and histologicevidence (using GFP and Ki67 immunostaining) of tumors, suggesting theywere disease-free after 210 days (FIG. 8 g ). Bdd is a versatiledelivery platform that can carry different chemotherapeutics. Aldox-Bddwas also able to prolong the animal survival compared to PBS and freeDOX (FIGS. 9 a-c ). However, it was not as effective as DM1-Bdd inimproving overall survival (FIG. 9 c ). Surprisingly, animals treatedwith free DOX had a shorter life expectancy compared to the PBS control,with all the animals continuously losing weight during DOX treatment(FIG. 9 d and e). They eventually died prior to receiving the finaldose, suggesting drug-induced toxicity and mortality.

Example 5: Anatomic Flexibility

DM1-Bdd for treating renal carcinoma was evaluated using a syngeneicmouse model for the studies, which involved a surgical implantation ofRenca cells (stably transduced with GFP and Luc) into the capsule of theright kidney of BALB/c mice (FIG. 5 a ). The tumor growth wasaggressive. A tumor mass of a substantial size developed as early as 1week after implantation (FIG. 5 b ). Imaging studies showed that i.v.DM1-Bdd was not only able to inhibit tumor progression (FIG. 5 c and d)but also reduced tumor size, as shown by decreased bioluminescencesignals in the majority of the animals during the treatment (FIG. 5 e ).There was 50% of survival after 160 days (FIG. 5 f and g). On the otherhand, all animals treated with either i.v. or i.t. DM1 died. In aseparate experiment, histological examination of the animals’ kidneysone week after completing the treatment course was performed (FIG. 5 h). Minimal tumor was present in animals treated with i.v. DM1-Bdd,whereas tumors of animals treated with DM1 or PBS control were large andaccompanied with an infiltration of mononuclear cells. Overall, the UDDapproach was anatomically flexible and could be used for treating tumorslocated at the upper urinary tract.

Example 6: Toxicity Profile

The toxicity profile of DM1-Bdd in healthy animals after completing 3weeks of weekly treatments was assessed. DM1-Bdd did not affect the redblood cell, leukocyte, or platelet counts or morphologic features (FIGS.6 a-b and FIG. 10 ). On the other hand, DM1 induced reticulocytosiswithout apparent anemia, and an inflammatory response characterized byincreased proportions of neutrophils, monocytes, and platelets. Thepositive control CIS was also toxic, as evidenced by thrombocytopeniaand lymphopenia. Serum biochemical analysis to assess for liver injuryand renal function were also performed. DM1-Bdd was not hepatotoxic,with no significant alteration in the release of ALP, ALT, and AST (FIG.6 c and FIG. 11 ). Importantly, the urea nitrogen/creatinine ratio wasnormal, suggesting that DM1-Bdd did not affect renal clearance ofnitrogenous waste. Histopathologic studies further confirmed that therewas no morphological evidence of injury in the liver, spleen, heart,lungs, or kidneys of the animals treated with DM1-Bdd (FIG. 6 d and FIG.12 ). In contrast, the biochemical testing showed that both DM1 and CISinduced hepatic and renal toxicity, as shown by increases in ALT and ASTactivities and BUN/creatinine ratios (FIG. 6 c ). They also inducedmuscle injury (CK activity), which would partly explain the increases inthe AST activity. Histologic examination also revealed that CIS causeddegeneration and necrosis of the proximal renal tubules, lunginflammation, and depletion of both erythrocytes and extramedullaryhematopoiesis (EMH) in spleen (FIG. 6 d and FIG. 12 ). Animals treatedwith DM1 showed similar renal damage, but to a lesser degree. Increasedsplenic and hepatic EMH with DM1 were also observed, which may explainthe observed reticulocytosis in the treated animals.

Discussion

Most chemotherapeutics, which are toxic to healthy and cancer cells, aregiven by infusion so that they can be continuously administered at ahigher total dose over a longer period of time. The goal is to achievemore effective treatment to improve patient tolerance to off-targettoxicities by maintaining the drug’s plasma concentration at a certainlevel and prolonging the tumor exposure to the drug. The present studyaimed to promote, rather than reduce, a drug’s clearance as anon-invasive alternative of ITC. Many bioactive peptides have beenapproved for treating various diseases, including cancer, diabetes, andcardiovascular disease. Without chemical modifications, peptides have ashort circulating half-life of several minutes. They are rapidlydegraded by protease enzymes and eliminated by renal filtration. In thepresent study it was recognized that a peptide’s rapid renal clearancecould be advantageous as a drug carrier to dispose most systemicallyadministered drugs in urine for treating NMIBC and reducing the unwantedsystemic side-effects. In the present invention a UDD approach wasintroduced by using a bio-inert, negatively charged peptide (Bdd) withminimal uptake by the recticuloendothelial system and other organs aswell as to be exclusively excreted into the urine. Bdd was employed fordelivering DM1, a microtubule inhibitor. DM1 was selected as it was100-fold more potent than commonly used ITC drugs, including MIT, DOXand CIS, against a panel of BC cell lines. Further, conjugating the drugto the Bdd peptide did not compromise cytotoxicity.

In terms of therapeutic efficacy, i.v.-administered peptide-DM1conjugate (DM1-Bdd) improved the overall survival in mice with BCcompared to conventional i.t. MIT (FIG. 4 h ). It was also moreeffective compared to the same treatment given by i.t. (FIGS. 4 f-i ).The improved efficacy was expected given that animals did not need tovoid shortly after i.v. injection (FIG. 4 a ). Promoting renal clearancecould reduce a drug’s off-target toxicity. DM1-Bdd did not induceunwanted toxicity (FIG. 6 ). In contrast, animals treated with DM1 orCIS showed evidences of hepatic and renal injury. The flexibility of UDDapproach in reducing drug-induced toxicity, by using aldox-Bdd for BCtreatment was also demonstrated. Unlike free DOX that caused mortality,improved survival was observed in those animals treated with aldox-Bdd(FIG. 9 ). However, compared to DM1-Bdd, the aldox-Bdd treatment onlyslowed down the cancer progression and did not eliminate tumors inindividual animals. The result was not unexpected since DM1 is morepotent than DOX (FIG. 3 c ).

ITC is a local treatment that only covers tumors in the bladder as thedrug solution cannot reach the upper urinary tract. DM1-Bdd isadministered through i.v. injection. This, together with the rapid renalclearance, should allow drugs to flush the entire URS. DM1-Bdd wasapplied for treating renal carcinomas and it was found that DM1-Bddoffered a significant survival benefit compared to the free drug. Infact, approximately 50% of the animals lack gross or histologic evidenceof the tumor one week after completing the treatment course. Intravenousdrug administration will thus allow more comprehensive coverage of theURS when used for treating BC, as tumors can extend, migrate into, orrecur throughout the entire urothelium, including the renal pelvis andthe ureter where 8-12% of the urothelial carcinomas originate from.Currently, when treating patients with renal pelvic or ureteral tumors,surgeons are often left with no choice but to remove the entire kidneyand ureter (even when the tumors are non-invasive) to prevent diseaserecurrence for URS tumors. DM1-Bdd can potentially be a kidney-sparingtreatment option for patients with upper tract urothelial cancers.

A drawback of ITC is the poor patient compliance rate (16-30%). Patientsreceiving ITC need to be catheterized weekly by trained personnel inhospital/clinic. In contrast, i.v. DM1-Bdd treatment is non-invasive,which can avoid complications associated with catheterization proceduresand improving patient’s quality of life and compliance. The lifetimemanagement of BC is costly, which requires repeated treatments becauseof its high recurrence rate. Systemic administration of achemotherapeutic, such as DM1-Bdd, will likely reduce hospitalizationcosts.

Overall, a UDD approach has been developed that could minimizenon-specific accumulation in other organs and offer a comprehensivetreatment by supplying drug to the entire URS, as a more effectivealternative to ITC. The developed DM1-Bdd is clinically translatable.The FDA has approved many peptides for treating different cancers. Theemployed DM1 is an active pharmacophore already used in antibody-drugconjugates, such as Herceptin-DM1 (T-DM1), for breast cancer treatment.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject disclosure have beendiscussed, the above specification is illustrative and not restrictive.Many variations of the disclosure will become apparent to those skilledin the art upon review of this specification and the claims below. Thefull scope of the disclosure should be determined by reference to theclaims, along with their full scope of equivalents, and thespecification, along with such variations.

We claim:
 1. A compound represented by formula (I):

or a pharmaceutically acceptable salt thereof, wherein the peptide is apeptide targeted for renal clearance.
 2. The compound of claim 1,wherein the peptide targeted for renal clearance comprises a sequence:

wherein: one of X, Y and Z is a β-amino acid residue; two of X, Y and Zare independently α-amino acid residues that each have at least one sidechain that comprises a carboxylic acid group; wherein each α-amino acidresidue is independently of D or L stereochemistry; m is from 2 to 10.3. The compound of any preceding claim, wherein the peptide has a zetapotential of from about -30 mV to about +20 mV at physiological pH. 4.The compound of claim 3, wherein the peptide has a zeta potential offrom about -20 mV to 0 mV at physiological pH.
 5. The compound of claim4, wherein the peptide has a zeta potential of from about -5 mV to 0 mVat physiological pH.
 6. The compound of any one of claims 2-5, whereinthe linker comprises one or more groups selected from: amide, imide,thiourea, thioether, disulfide, alkyl, aryl, polyether, hydrazone,ester, carbonate, ketal and silyl ether.
 7. The compound of any one ofclaims 2-6, wherein the active moiety is a therapeutic agent or animaging agent.
 8. The compound of any preceding claim, wherein: thepeptide targeted for renal clearance comprises a sequence:

wherein: one of X, Y and Z is a β-amino acid residue; two of X, Y and Zare independently α-amino acid residues that each have at least one sidechain that comprises a carboxylic acid group; wherein each α-amino acidresidue may independently be of D or L stereochemistry; m is from 2 to10; the linker comprises one or more groups selected from: amide, imide,thiourea, thioether, disulfide, alkyl, aryl, polyether, hydrazone,ester, carbonate, ketal and silyl ether; and the active moiety is atherapeutic agent or an imaging agent.
 9. The compound of any precedingclaim represented by formula (IA):

.
 10. The compound of any one of claims 2-9, wherein the β-amino acidresidue does not comprise an ionizable side chain.
 11. The compound ofany one of claims 2-10, wherein the β-amino acid residue is a β-alanineresidue.
 12. The compound of any one of claims 2-11, wherein X is aβ-alanine residue.
 13. The compound of claim 2, wherein each α-aminoacid residue is independently selected from an aspartic acid residue anda glutamic acid residue.
 14. The compound of claim 2, wherein at leastone α-amino acid residue is an unnatural amino acid residue.
 15. Thecompound of claim 14, wherein the unnatural α-amino acid residue has atleast two side chain carboxylic acid groups.
 16. The compound of claim15, wherein the unnatural α-amino acid residue is selected from a2-aminoethane-1,1,2-tricarboxylic acid residue and a2-aminopropane-1,2,3-tricarboxylic acid residue.
 17. The compound ofclaim 13, wherein each Y and Z are aspartic acid residues.
 18. Thecompound of claim 17, wherein each Y and Z are D-aspartic acid residues.19. The compound of claim 2, wherein X, Y and Z are each independentlyselected from a β-alanine residue, an aspartic acid residue and aglutamic acid residue.
 20. The compound of any one of claims 2-19,wherein m is
 4. 21. The compound of any one of claims 2-20, wherein thelinker comprises a group selected from:

.
 22. The compound of any one of claims 2-21, wherein the linkercomprises a group derived from N-succinimidyl3-(2-pyridyldithio)propionate or succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate.
 23. The compound of anypreceding claim, wherein the active moiety is a therapeutic agent. 24.The compound of any preceding claim, wherein the therapeutic agent isselected from an anticancer agent, an antibiotic, an agent that treatsoveractive bladder, an agent that treats urinary incontinence, an agentthat treats interstitial cystitis and an agent that treats kidneystones.
 25. The compound of claim 23, wherein the therapeutic agent isselected from 13-cis-Retinoic Acid, 2-Chlorodeoxyadenosine,5-Azacitidine, 5-Fluorouracil, 6-Mercaptopurine, 6-Thioguanine,actinomycin-D, adriamycin, aldesleukin, alemtuzumab, alitretinoin,all-transretinoic acid, alpha interferon, altretamine, amethopterin,amifostine, anagrelide, anastrozole, arabinosylcytosine, arsenictrioxide, amsacrine, aminocamptothecin, aminoglutethimide, asparaginase,azacytidine, bacillus calmette-guerin (BCG), bendamustine, bevacizumab,bexarotene, bicalutamide, bortezomib, bleomycin, busulfan, calciumleucovorin, citrovorum factor, capecitabine, canertinib, carboplatin,carmustine, cetuximab, chlorambucil, cisplatin, cladribine, cortisone,cyclophosphamide, cytarabine, darbepoetin alfa, dasatinib, daunomycin,decitabine, denileukin diftitox, dexamethasone, dexasone, dexrazoxane,dactinomycin, daunorubicin, decarbazine, docetaxel, doxorubicin, doxil,aldoxorubicin, doxifluridine, edrecolomab, eniluracil, epirubicin,epoetin alfa, erlotinib, everolimus, exemestane, estramustine,etoposide, filgrastim, fluoxymesterone, fulvestrant, flavopiridol,floxuridine, fludarabine, fluorouracil, flutamide, gefitinib,gemcitabine, gemtuzumab ozogamicin, goserelin, granulocyte-colonystimulating factor, granulocyte macrophage-colony stimulating factor,hexamethylmelamine, hydrocortisone hydroxyurea, ibritumomab, ibritumomabtiuxetan, interferon alpha, interleukin-2, interleukin-11, isotretinoin,ixabepilone, idarubicin, imatinib mesylate, ifosfamide, irinotecan,lapatinib, lenalidomide, letrozole, leucovorin, leuprolide, liposomalAra-C, lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine,mertansine, mesna, methotrexate, methylprednisolone, mitomycin C,mitotane, mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin,oxaliplatin, paclitaxel, pamidronate, pemetrexed, panitumumab, PEGInterferon, pegaspargase, pegfilgrastim, PEG- L-asparaginase,pentostatin, plicamycin, prednisolone, prednisone, procarbazine,raloxifene, rituximab, romiplostim, ralitrexed, sapacitabine,sargramostim, satraplatin, sorafenib, sunitinib, semustine,streptozocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus,temozolamide, teniposide, thalidomide, thioguanine, thiotepa, topotecan,toremifene, tositumomab, trastuzumab, trastuzumab emtansine, tretinoin,trimitrexate, alrubicin, vincristine, vinblastine, vindestine,vinorelbine, vorinostat, and zoledronic acid.
 26. The compound of anyone of claims 2-25, wherein the therapeutic agent is an anticanceragent.
 27. The compound of claim 26, wherein the anticancer agent isselected from mertansine, doxorubicin, dasatinib, cisplatin, mitomycin,gemcitabine and paclitaxel.
 28. The compound of claim 2, wherein, X is aβ-alanine residue; and Y and Z are D-aspartic acid residues.
 29. Thecompound of claim 2, wherein, X is a β-alanine residue; Y and Z areD-aspartic acid residues; and m is
 4. 30. The compound of claim 2,wherein, X is a β-alanine residue; Y and Z are D-aspartic acid residues;m is 4; the linker comprises a disulfide group; and the active moiety ismertansine.
 31. The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt thereof, wherein B is a β-alanineresidue and D is an aspartic acid residue of D or L configuration. 32.The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt thereof, wherein B is a β-alanineresidue and D is an aspartic acid residue of D or L configuration.
 33. Apharmaceutical composition comprising a compound of any preceding claim.34. The pharmaceutical composition of claim 33, wherein the compositionis formulated for intravenous administration.
 35. A method of treating acancer, a urinary tract infection, overactive bladder, urinaryincontinence, interstitial cystitis or kidney stones comprisingadministering to a patient in need thereof a compound or composition ofany preceding claim.
 36. A method of treating cancer, comprisingadministering to a patient in need thereof a compound or composition ofany preceding claim.
 37. The method of claim 36, wherein the cancer is acancer of the kidney or urinary tract.
 38. The method of claim 37,wherein the cancer is bladder cancer.
 39. The method of claim 38,wherein the bladder cancer is non-muscle invasive bladder cancer. 40.The method of claim 39, wherein the bladder cancer is urothelialcarcinoma.
 41. The method of any one of claims 35-40, wherein thecompound is administered intravenously.